This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Sortase mediated pili assembly is the hallmark of Gram-positive pili biogenesis. The pilus specific sortases and housekeeping sortase are known to perform distinct functions of pili assembly and anchoring, respectively, however, the structural basis of such enzyme mediated pili biogenesis has not yet been well understood. The PI-1 pathogenicity island of Streptococcus agalactiae, a major cause of infections in neonates and immune-compromised elderly, is one of the simplest pili systems that encodes one housekeeping sortase (sortase A) and two pili-specific sortases (sortases C1 and C2). We are investigating the structural basis for the observed narrow substrate specificity and efficiency of both classes of these enzymes. We have crystallized S. agalactiae sortase A, and sortase C1 and an inhibitor complex of the later. We would like to delineate the unique structural features and similarities between the two classes of sortases.